Nickel column chromatography

Wt nuclear dUTPase and mutants were expressed in E. coli BL21(DE3)RIPL (New England Biolabs Inc., Beverly, MA) for 1.5 h at 37°C using 1 mM isopropyl beta‐d‐1‐thiogalactopyranoside (IPTG). The cells were harvested at 6000g for 20 min at 4°C. The pellet was washed with 10 mL of PBS then frozen at −80°C overnight. The cells were suspended in Bugbuster reagent (EMD Millipore, Billerica, MA) where the standard company protocol was followed. Nickel column chromatography (Qiagen, Valencia, CA) was used for purification following standard company protocol. Briefly, the column was equilibrated and washed with “Buffer 1” consisting of 50 mM sodium phosphate pH 7.4, 300 mM NaCl, and 10 mM imidazole. The protein was eluted with “Buffer 2″ containing 50 mM sodium phosphate pH 7.4, 300 mM NaCl, and 150 mM imidazole. The eluted fractions containing the purified protein were pooled and then subjected to overnight dialysis against 50 mM Tris–HCl pH 7.5, 50 mM NaCl, and 10% glycerol. The protein, if needed, was concentrated using Amicon Ultra115 centrifugal filter (UFC901008) using the protocol of the manufacturer.